Strategies to repair injured hearts have predominantly focused on infusion, direct injection, or transplantation of any of a variety of types of stem/progenitor cells. We have recently reported that conditional deletion of GSK-3b specifically in cardiomyocytes leads to proliferation of cardiomyocytes. Furthermore, we find that the degree of proliferation is amplified in the setting of stress induced by thoracic aortic constriction (producing marked increases in left ventricular systolic pressure) or myocardial infarction (MI). In fact, we have found that deletion (or inhibition) of GSK-3b leads to proliferation of cardiomyocytes in mouse embryonic stem cell-derived embryoid bodies, in neonatal rat ventricular myocytes in culture, in cardiomyocytes in the developing heart, in cardiomyocytes in the normal heart, and, as noted above, particularly in cardiomyocytes in the stressed heart. If this proliferation is sufficiently robust, one could induce meaningful regeneration without the need for transplantation (i.e. regeneration in situ). We will address this hypothesis with three Specific Aims. We will first determine how robust is the cardiomyocyte proliferation resulting from deletion of GSK-3b by quantifying proliferation rates and cardiomyocyte mass in both the normal heart and in the stressed heart. We will then examine the physiologic consequences of this proliferation by determining whether there is a beneficial effect on left ventricular function. Finally, we will determine whether deletion of GSK-3b can lead to beneficial effects in mice with moderately advanced heart failure- recapitulating the clinical scenario of patients with advanced heart failure for whom there are very few treatment options besides heart transplant or destination LVAD therapy. In a future directions section, we also discuss the critical role played by GSK-3b in driving differentiation of immature cardiomyocytes into mature cardiomyocytes. We propose studies to restore expression of GSK-3b via adeno-associated virus gene transfer to the heart after the proliferative response has created new myocytes. Thus any immature myocytes formed in response to the deletion of GSK-3b will be differentiated into mature, fully functional myocytes, maximizing the chances of improving LV function. We believe that this application perfectly matches the goals of the R21 mechanism of supporting groundbreaking research with the potential to significantly advance therapeutic options for heart failure patients- a disease that has had no new important classes of agents come to market in many years. PUBLIC HEALTH RELEVANCE: Herein we propose to determine whether it is possible to drive cardiomyocyte proliferation sufficiently to result in cardiac regeneration in situ (i.e. without the need to utilize exogenous stem/progenitor cells). Based on our extensive studies in mouse models, we hypothesize that by manipulating a single signaling pathway- the glycogen synthase kinase-3b (GSK-3b) signaling pathway- in vivo, we will indeed be able to achieve regeneration. If we can achieve this, it would be a significant step forward in the treatment of patients with end- stage heart diseases of many etiologies.